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Antibiotic Resistance, Pathogenicity Factors and Bacteriocin Production in Enterococci Recovered from Fresh Vegetables and Farm-associated Samples in South Korea

국내 신선 채소와 농장 관련 시료에서 분리한 장구균의 항생제 내성, 병원성 인자, 박테리오신 생성에 관한 연구

초록/요약

Enterococci are Gram-positive bacteria normally found in a variety of foods such as cheeses, vegetables and fermented foods due to their potential benefits. While much resistance and virulence have been reported in enterococci from clinical areas, few were done in fresh vegetables and farm-related samples in South Korea. Thus, Chapter 1 investigated the antibiotic resistance patterns and pathogenicity factors associated with their virulence traits. 220 Enterococcus isolates (106 Enterococcus faecalis, 55 Enterococcus faecium, 27 Enterococcus casseliflavus, 21 Enterococcus gallinarum, 8 Enterococcus hirae, 2 Enterococcus durans and 1 Enterococcus cecorum) were tested against thirteen antibiotics by disk diffusion method and subjected to β-hemolysis, gelatinase assay and the presence of virulence genes. Most isolates were resistant to rifampicin (61.8%), quinupristin/dalfopristin (56.3%), streptomycin (53.1%) and ciprofloxacin (48.6%). 23.1% of isolates were resistant to five or more antibiotics. One E. faecalis (EFS16-ER-RD15) from stream sample was able to demonstrate β-hemolysis activity and 47.2% were able to hydrolyze gelatin. 58.6% carried one or more virulence genes with gelE genes were primarily detected (49.0%). On the other hand, strain-typing analyses by DiversiLab® revealed a large proportion of isolates with 100% genetically similar. Chapter 2 aimed to identify the minimal inhibitory concentrations (MICs) and to check for rifampicin and ciprofloxacin genes in addition to investigate the effect of efflux pump inhibitor using phenylalanine-arginine β-naphthylamide (PAβN) on rifampicin and ciprofloxacin-resistant enterococci based on disk diffusion results previously. 53.6% rifampicin-resistant isolates indicated MIC ≥4 μg/ml while 41.1% of ciprofloxacin resistant isolates showed MIC ≥4 μg/ml. PCR for rifampicin and ciprofloxacin genes indicated that rpoB4 and gyrA genes were primarily detected, respectively. Following the treatment with 20 μg/ml PAβN, 57.5% of rifampicin-resistant isolates and 63.6% of ciprofloxacin-resistant isolates exhibited reductions while the rest displayed no shift in MICs. Meanwhile, Chapter 3 investigated the bacteriocinogenic activities of these enterococci against fourteen food poisoning and indicator bacteria in addition to check for the presence of bacteriocin and cytolysin genes and the resistance patterns of these isolates. 45.9% displayed bacteriocin activity with major inhibitions were seen against Listeria monocytogenes ATCC 19118 (24.0%). 79 isolates showed at least the presence of one or more bacteriocin genes with entA was predominantly detected (63.3%). Two E. faecium isolates (EFM01-WA-RD14 and EFM49-WG-RD15) originated from work area and work gloves respectively carried up to four genes. All isolates were γ-hemolysis (non-hemolysis). All isolates were fully susceptible to penicillin (100.0%) and vancomycin (100.0%). It was found that five isolates (two E. faecium and three E. casseliflavus) fit the traits as potential bacteriocin producers. In conclusion, this study provides an insight into the diversity of antibiotic resistance, virulence as well as their potential ability to secrete bacteriocins from samples not frequently studied. These results are significant considering that the enterococci were isolated from fresh vegetables and farm-related samples covering eight provinces in South Korea. Further studies are necessary to fully understand the prospective of these enterococci especially those strains, which have the potential effects against foodborne pathogens.

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목차

CHAPTER 1 Antibiotic Resistant and Pathogenicity Factors of Enterococci Isolated from Fresh Vegetables and Farm-associated Samples in South Korea. 1
Abstract. 2
1.1 Introduction 3
1.2 Materials and Methods. 6
1.2.1 Strains 6
1.2.2 Antimicrobial susceptibility testing 7
1.2.3 Hemolysis and gelatinase testing 8
1.2.4 Detection of virulence genes by PCR. 8
1.2.5 Genetic strain typing using DiversiLab system. 9
1.3 Results 10
1.3.1 Enterococcal isolates. 10
1.3.2 Antimicrobial susceptibility testing 10
1.3.3 Minimal inhibitory concentration (MIC) 11
1.3.4 Hemolytic activity. 11
1.3.5 Gelatinase activity. 12
1.3.6 Detection of virulence factors. 12
1.3.7 Comparison between phenotypic and molecular screening 13
1.3.7.1 Presence of gelE gene and gelatinase production 13
1.3.7.2 Presence of cytolysin genes (cylA, cylB, cylM) and beta hemolytic activity. 14
1.3.8 Genetic strain typing using DiversiLab system. 15
1.4 Discussion 17
References. 29
Tables
Table 1.1 The oligonucleotide primer pairs used for the detection of virulence genes. 39
Table 1.2 Distribution of enterococci isolates from vegetables and farm-associated samples in South Korea 40
Table 1.3 Antimicrobial resistance patterns of enterococci (CLSI, 2014) 41
Table 1.4 Resistance patterns of enterococci by MIC method (CLSI, 2014) 43
Table 1.5 Distribution of virulence genes in enterococci isolates 44
Table 1.6 Enterococci isolates and their respective origins, which displayed
100% genetic similarities in DiversiLab system 46
Figures
Figure 1.1 Distribution of gelatinase producers and gelE genes among all Enterococcus species 45
Figure 1.2 Dendrogram analyses of enterococci isolates. Boxes indicate 100% genetic similarities between isolates 56
CHAPTER 2 Influence of Efflux Pump Inhibitor on Rifampicin and Ciprofloxacin Resistant Enterococci Isolated from Vegetables and Environmental Samples in South Korea 57
Abstract. 58
2.1 Introduction. 59
2.2 Materials and Methods 63
2.2.1 Strains. 63
2.2.2 Determination of minimum inhibitory concentration (MIC) of rifampicin and ciprofloxacin-resistant isolates. 63
2.2.3 Efflux pump inhibition test using phenylalanine-arginine β-naphthylamide (PAβN). 64
2.2.4 Detection of rifampicin and ciprofloxacin resistance genes. 64
2.3 Results 66
2.3.1 Strains. 66
2.3.2 Determination of minimum inhibitory concentration (MIC) of rifampicin and ciprofloxacin-resistant isolates. 66
2.3.3 Detection of rifampicin and ciprofloxacin resistance genes. 67
2.3.4 Efflux pump inhibition test using phenylalanine-arginine β-naphthylamide (PAβN). 68
2.4 Discussion. 70
References. 76
Tables
Table 2.1 Primers used for detection of rifampicin and ciprofloxacin resistance genes 82
Table 2.2 Distribution of rifampicin-resistant genes in rifampicin resistant isolates. 83
Table 2.3 Distribution of ciprofloxacin-resistant genes in ciprofloxacin resistant isolates. 84
Table 2.4 Resistance profile and characteristics of rifampicin and ciprofloxacin resistance in enterococci isolates 85
CHAPTER 3 Characterization of Bacteriocin-Producing Enterococci Isolated from Vegetables and Farm-related Samples in South Korea. 86
Abstract. 87
3.1 Introduction. 88
3.2 Materials and Methods 93
3.2.1 Strains. 93
3.2.2 Detection of bacteriocin production using spot agar method. 93
3.2.3 Hemolytic activity 94
3.2.4 DNA extraction 94
3.2.5 PCR detection for bacteriocin and cytolysin genes 95
3.2.6 Antimicrobial susceptibility testing . 96
3.3 Results. 97
3.3.1 Detection of bacteriocin production using spot agar method. 97
3.3.2 Hemolytic activity and cytolysin genes 99
3.3.3 Detection of bacteriocin genes 100
3.3.4 Antimicrobial susceptibility testing 101
3.4 Discussion 103
References. 109
Tables
Table 3.1 PCR primers and product sizes for detection of enterocin and cytolysin genes 117
Table 3.2 Bacteriocin genes distribution among 101 bacteriocin producers. 120
Table 3.3 Antimicrobial susceptibility patterns for bacteriocin-producing enterococci. 121
Table 3.4 Characteristics of five enterococci, (two E. faecium and three E. casseliflavus) which were susceptible to all the tested antibiotics 122
Table 3.5 Food poisoning and indicator bacteria used for bacteriocin screening of enterococci . 123
Figures
Figure 3.1. The effects of bacteriocin-producing enterococci against food poisoning and indicator bacteria (ATCC group) 118
Figure 3.2. The effects of bacteriocin-producing enterococci against food poisoning and indicator bacteria (non-ATCC group) 119
ABSTRACT IN KOREAN (국문초록) 124
ACKNOWLEDGEMENTS 128

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